Data on exopolysaccharides produced by Bacillus spp. from cassava pulp with antioxidant and antimicrobial properties

This data evaluated the capacity of Bacillus spp. isolated from Thai milk kefir to produce exopolysaccharide (EPS) on cassava pulp and tested its antioxidant and antibacterial properties. Thailand's starch industry generates million tons of cassava pulp, which is underutilized or bio-transformed into higher-value bioproducts. Antioxidant and antibacterial bacterial exopolysaccharides are beneficial in the food, feed, pharmaceutical, and cosmetic industries. Moisture, ash, fat, protein, fiber, starch, sugar, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were analyzed from cassava pulp as an EPS substrate. After 3 days of bacterial fermentation, EPS generation, culture pH, reducing sugar amount, and bacterial count were recorded. Antioxidant activities and bioactive content including hydroxyl radical scavenging activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP), total phenolic and flavonoid content (TPC and TFC), and antimicrobial activity against two Nile tilapia pathogens (Streptococcus agalactiae and Staphylococcus aureus) from different Bacillus species were evaluated. Proximate analysis, dinitrosalicylic acid assay, pH value record, bacterial count using spread plate method, antioxidant activity and bioactive content assays via spectrophotometry, and agar disk diffusion were the main approaches. This study used microbial cell factories to convert agro-biowaste, such as cassava pulp, into EPS bioproducts which accords with a bio-circular green economy model.


a b s t r a c t
This data evaluated the capacity of Bacillus spp. isolated from Thai milk kefir to produce exopolysaccharide (EPS) on cassava pulp and tested its antioxidant and antibacterial properties. Thailand's starch industry generates million tons of cassava pulp, which is underutilized or biotransformed into higher-value bioproducts. Antioxidant and antibacterial bacterial exopolysaccharides are beneficial in the food, feed, pharmaceutical, and cosmetic industries. Moisture, ash, fat, protein, fiber, starch, sugar, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were analyzed from cassava pulp as an EPS substrate. After 3 days of bacterial fermentation, EPS generation, culture pH, reducing sugar amount, and bacterial count were recorded. Antioxidant activities and bioactive content including hydroxyl radical scavenging activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP), total phe-nolic and flavonoid content (TPC and TFC), and antimicrobial activity against two Nile tilapia pathogens ( Streptococcus agalactiae and Staphylococcus aureus ) from different Bacillus species were evaluated. Proximate analysis, dinitrosalicylic acid assay, pH value record, bacterial count using spread plate method, antioxidant activity and bioactive content assays via spectrophotometry, and agar disk diffusion were the main approaches. This study used microbial cell factories to convert agro-biowaste, such as cassava pulp, into EPS bioproducts which accords with a bio-circular green economy model.

Value of the Data
• This work presented the raw data obtained during the 3-day fermentation of EPS production using cassava pulp as substrate by eight different Bacillus spp. The data consisted of the EPSproducing content of each strain and its bioactivities. • The dataset would provide insight into selecting the bacterial strain and using cassava pulp as substrate to generate a high EPS content with antioxidant activity and antimicrobial properties against two bacterial pathogens of Nile tilapia. • The dataset is useful for agriculturists, researchers, and the private sector who wish to valorize cassava pulp via bacterial fermentation to produce bioactive EPS products. • It is anticipated that the applicability of the dataset will allow for the commercialization of crude EPS bioproducts as feed additives. • This work proposes an alternative, low-cost means of producing value-added EPS bioproducts from other agro-biowastes in addition to cassava pulp via bacterial fermentation, thereby enabling a bio-circular green economy.

Objective
This work aimed to determine the bioactivities of EPSs produced from cassava pulp (agroindustry-sourced biowaste) as substrate via a 3-day fermentation by different Bacillus spp.

Data Description
Chemical composition analysis of cassava pulp showed the predominance of starch and sugar at 64.12% followed by neutral detergent fiber (NDF) at 33.22%, acid detergent fiber (ADF) at 24.32%, fiber at 17.56% and protein at 12.09% ( Table 1 ). The compositions of ash, fat, protein and acid detergent lignin (ADL) were all below 5%. This data is useful when it comes to considering the influence of chemical composition of substrate on EPS synthesis by Bacillus spp. Eight EPS-producing bacteria from the Bacillus genus isolated from Thai milk kefir were tested in this work [1] . Bacterial EPS production from cassava pulp by Bacillus spp. over 3 days was shown in Fig. 1 . On day 0, the medium containing cassava pulp substrate appeared brownish ( Fig. 1 A) ; however, over 3 days of bacterial fermentation, the culture appeared yellowish ( Fig. 1 B-1 I). The crude EPS products from each strain looked similar, with whitish and creamy features . Different Bacillus bacteria produced EPS differently. B. tequilensis PS21 produced the highest EPS content of 11.38 g FW/100 mL and 1.49 g DW/100 mL ( Table 2 ). The pH values of the media in all strains dropped slightly from initially 7.0 (day 0) to about 6.31-6.44 over 3 days ( Table 2 ). Similarly, the total plate count (TPC) of bacteria was similar (log 8.82-9.00 CFU/mL on day 3) in all strains. Antioxidant activities and bioactive contents of bacterial EPSs from cassava pulp are shown in Table 3 . The dextran (20 mg/mL) EPS standard exhibited the most potent 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging activity of 73.98%, a ferric reducing antioxidant power (FRAP) value of 2.23 mg Fe 2 + /g DW, and hydroxyl radical scavenging activity of 80.94%. Amongst eight strains, EPS produced by B. tequilensis PS21 displayed the strongest DPPH radical scaveng- ing activity of 43.93%, a FRAP value of 0.71 mg Fe 2 + /g DW, and a hydroxyl radical scavenging activity of 44.26% ( Table 3 ). Likewise, B. tequilensis PS21 displayed the highest TPC of 6.31 mg GAE/g DW. Antibacterial activities of EPSs by eight Bacillus spp. on two common bacterial pathogens that cause infection in Nile tilapia, S. agalactiae and S. aureus , using an agar disk diffusion method are shown in Fig. 2 and Table 4 .  Penicillin antibiotics (10 μg/mL disk) as a positive control showed the highest clear zones on both pathogens, followed by dextran (20 mg/mL) as an EPS standard against S. agalactiae ( Table 4 and Fig. 2 A). However, dextran was not that effective against S. aureus ( Fig. 2 B).

Cassava Pulp Substrate Preparation
The cassava pulp was ground into a powder by milling and sieving, and the resulting powder was stored in a desiccator until it was required [2] .

Chemical Composition Analysis
Proximate analysis according to standard AOAC procedures [3] were used to determine the levels of ash, moisture, and crude fat. The percentages of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) were calculated in the same manner as previously published [4] . The amount of crude protein was determined using the Kjeldhal technique.

Bacterial Sources
Eight EPS-producing bacteria from the Bacillus genus isolated from Thai milk kefir of Kamphaeng Phet Province, Thailand [1] were used for EPS production using cassava pulp as substrate.

Preparation of Cassava Pulp for Bacterial EPS Production
Cassava pulp powder at 5% (w/v) was mixed in autoclaved 100 mL distilled water. This was used as the medium for bacterial EPS production. The inoculum of overnight bacterial culture at 3% (v/v) was inoculated into the media and aerobically incubated for 3 days at 37 °C with agitation at 150 rpm. The reducing sugar content and pH values were recorded daily for 3 days using the DNS method [5] and a pH meter (FiveEasy Plus pH meter FP20, Port Melbourne, Australia). Glucose standard and DNS were purchased from Sigma-Aldrich, MO, USA. Total plate count (TPC) for bacterial growth at day 1-3 was conducted using the spread plate technique and serial dilutions. EPS quantity was determined on day 3.

Extraction of Crude EPS
After 3 days, the bacterial culture was spun at 16,100 g at 4 °C for 30 min in a centrifuge (Hettich® Universal 320/320R centrifuge, Kirchlengern, Germany). Two hundred milliliters of cold absolute ethanol (RCI Labscan Limited, Bangkok, Thailand) were mixed into the supernatant at 4 °C for 24 h without shaking. After centrifugation, EPS precipitate was rinsed twice with 100% ethanol. Fresh EPS was weighed using a fine balance (Presica 25A, Switzerland) and dried crude EPS was obtained as previously described [6] . Next, dried crude EPS (20 mg) was resuspended in 1 mL of sterile distilled water, filtered through 0.20 μm membrane filter (Sartorius Minisart TM High Flow Syringe Filters, Thermo Fisher Scientific Inc., MA, USA) and stored at −20 °C until use.

Scavenging activity
where A blank contained deionized water, A control contained all reagents without the EPS sample, and A sample contained all reagents and EPS sample. Dextran was used as an EPS standard. All chemicals were from Sigma-Aldrich, MO, USA.

DPPH Radical Scavenging Activity
In a test tube, 2.0 mL of water, 1 mL of EPS sample (20 mg/mL), and 0.4 mL of DPPH ethanolic solution (0.5 mM) were mixed [6] . Thirty minutes were spent incubating the containers at 30 ± 3 °C in the dark. At 517 nm, the absorbance was recorded using M965 + microplate reader (MeterTech, Taipei, Taiwan).

Scavenging activity
where A blank contained deionized water, A control contained all reagents without the EPS sample, and A sample contained all reagents and EPS sample. Dextran was used as an EPS standard. All chemicals were from Sigma-Aldrich, MO, USA.

TPC
This was analyzed in the same way as was explained in the previous work [9] . A total volume of 100 μL of 10% Folin-Ciocalteu solution was mixed with 20 μL of EPS (20 mg/mL), 80 μL of 7.35% sodium carbonate. After a 30-min dark response, a record at A 765nm was taken using M965 + microplate reader (MeterTech, Taipei, Taiwan). Gallic acid served as a standard. All chemicals were from Sigma-Aldrich, MO, USA.

Antimicrobial Activity
An agar disk diffusion test was carried out. S. agalactiae and S. aureus , the most common infectious agents in Nile tilapia, were employed to test EPSs' antibacterial efficacy. S. agalactiae EW1 (GenBank accession no. OR272051.1) was isolated from diseased Nile tilapia in the northeastern region of Thailand [10] . S. aureus TISTR 517 (ATCC 25923) (GenBank accession no. OP522324.1) was obtained from Thailand Institute of Scientific and Technological Research (TISTR), Thailand. Both bacteria were grown for 24 h at 37 °C in Luria-Bertani (LB) broth (HiMedia, Maharashtra, India), and the cultures were adjusted to 10 8 CFU/mL determined by A 600nm . Paper discs (4 mm diameter) were placed upon LB agar plates inoculated with 100μL of bacterial suspension. Each sample disk contained 20 μL of sterilized EPS (20 mg/mL). LB agar dishes were incubated at 37 °C for 48 h. The antimicrobial activity was assessed as the record of the inhibition zone diameter in mm. Dextran (20 mg/mL) was used as an EPS standard, and penicillin (10 μg/mL) from Sigma-Aldrich, MO, USA was utilized as a positive control.

Statistical Analysis
Triplicates were used for each treatment, and mean ± SD was reported. SPSS Demon version (IBM, Armonk, NY, USA) was used to assess significant mean differences using ANOVA and Duncan's multiple range test at p < 0.05.

Ethics Statements
This article does not contain any dataset involving animals or human participants performed by any of the authors.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
Raw dataset for bacterial exopolysaccharide production using cassava pulp as substrate by Bacillus spp. isolated from Thai milk kefir with its antioxidant and antimicrobial capacities (Original data) (Mendeley Data).